The invention provides new uses and compositions of proteoglycans. The proteoglycans are derived from perlecan, an extracellular matrix protein. The inventive proteoglycans retain certain desirable activities of the full-length perlecan molecule, such as the ability to bind growth factors, yet they have a size that allows for effective preparation and application as is not the case for perlecan. Furthermore, large amounts of the inventive proteoglycan can be produced in mammalian cell lines. The inventive proteoglycans can be used as adhesive coatings on scaffolds used for bone and tissue repair to attract and retain growth factors to the repair site. The inventive proteoglycans can also be used to induce differentiation to or maintenance of chondrocyte phenotype.
Chondrogenesis occurs as a multi-step process that is initiated by condensation of mesenchymal stem cells that subsequently undergo a specific program of differentiation. Studies from several laboratories clearly have demonstrated a role for specific soluble signals in this differentiation program that include bone morphogenetic proteins (1), parathyroid hormone related protein (PTIrP)(2), Indian hedgehog (Ihh) (3), and transforming and fibroblast growth factors (4,5). Of interest, several of these are known to interact with heparan sulfate proteoglycans (HSPG), a factor implicated in modulating their bioavailability (6). It has been demonstrated that a large HSPG found in the extracellular matrix (ECM) of developing cartilage, perlecan (Pln, HSPG2), stimulated cells of a murine fibroblast line, C3H10T1/2, to form aggregates in vitro similar to those found in condensing mesenchyme in vivo (7). In addition, Pln maintained the chondrogenic phenotype of adult chonodrocytes in vitro (7).
Consistent with a fundamental role for Pln in endochondral bone formation, targeted disruption of the Pln gene in mice results in severe disorganization of the columnar structure of chondrocytes and defective endochondral ossification (9). Interestingly, the phenotype of the Pln null mice is similar to that caused by activating mutations of fibroblast growth factor receptor 3 (FGFR3), which has been interpreted to mean that these molecules modulate similar signaling pathways in developing cartilage (9).
Pln is a multi-domain protein consisting of five distinct regions, four of which display sequence similarity to other protein families (10). The proteoglycan and its core protein are disclosed in Costell et al. (16). All the perlecan domains are disclosed in Noonan DM et al. (10).
The N-terminal domain I is unique to Pln. Within domain I are three glycosaminoglycan (“GAG”) attachment sites, defined by the consensus amino acid triplet Serine- Glycine-Aspartic Acid (“SGD”). While other potential sites for glycosylation exist in the protein core, the N-terminal sites are considered the major site for GAG attachment (11). Domain II contains repeat sequences highly similar to domain IV of the laminin A chain. In mice, domain III contains an Arginine-Glycine-Aspartic acid (“RGD”) sequence but in human Pln this sequence is missing (12). Domain IV contains repeats similar to those found in the IgG superfamily member, neural cell adhesion molecule (N-CAM). The C-terminal of domain V shows sequence similarity to G region of the laminin A. chain. There are also epidermal growth factor (EGF)-like sequences spaced between the domain G-like repeats in Pln domain V.
Each domain of Pln previously has been produced as a recombinant protein, and several of these also have been produced in various forms (13-17).
Perlecan has been associated with growth factors. Mongiat et al. reported that perlecan acts as a ligand reservoir for various growth factors, stabilizing them against misfolding and proteolysis (20). Costell et al. reported that perlecan binds and delivers growth factors in two ways (8). Costell et al. reported that perlecan's heparin sulfate and chondroitin sulfate side chains bind growth factors as well as its protein core.
Several groups have studied the interaction between the glycosaminoglycan molecules and the fibroblast growth factor family of heparin-binding growth factors. For example, Walz et al. have found that the biological activities of fibroblast growth factor-1 and fibroblast growth factor-2 depend on their ability to bind cell surfaces and extracellular matrix heparin sulfate side chains, like those found attached to perlecan (27).
Growth factors have been used as coatings for scaffolds implanted to treat numerous skeletal and connective-tissue related disorders. It is of great interest to attract and retain growth factors to the site of bone or tissue repair and thereby accelerate healing.
Although it is known that perlecan is involved in growth factor retention, the intact molecule is too large to exploit commercially as a growth factor adhesive. Perlecan is one of the most complex gene products because of its enormous dimensions and number of posttranslational modifications. Its size does not allow for efficient and cost effective commercial production. The present invention avoids this problem and meets the needs of the art by providing a molecule that can be produced in large amounts in mammalian cell lines and is at least as active as the intact perlecan molecule in binding and presenting heparin-binding growth factors and inducing differentiation to or maintenance of chondrocyte phenotype.